Characterization of a Microchip Flow-Chamber System for the Analysis of Platelet Thrombus Formation Using Whole Blood Samples - Studies On Healthy Subjects

A flow-chamber system was developed to evaluate the growth of platelet thrombus formation (PTF) quantitatively using whole blood under various shear stress conditions. This device, T-TAS (Total Thrombus-formation Analysis System, Fujimori Kogyo Co., Yokohama, Kanagawa), analyzes the process of PTF by monitoring the continuous pressure increase in the capillary of microchip where whole blood flows, using two kinds of thrombogenic surfaces (PL chip: coated with collagen, AR chip: coated with collagen plus tissue factor). In the current study, we characterized this system using whole blood samples from healthy subjects by comparing the measurements with those of other standard platelet function tests.

Whole blood samples were collected from 32 healthy volunteers with hirudin (PL chip) or 3.2% sodium citrate (AR chip) as anticoagulants. For AR chip, CaCl₂ with corn trypsin inhibitor was mixed immediately before the testing. The samples were individually applied on the system to measure the PTF starting time (T₁₀: time to reach 10 kPa), occlusion time (OT: T₆₀, time to reach 60 kPa for PL chip; T₈₀, 80 kPa for AR chip), and AUC (area under the flow pressure curve: AUC₁₀, until 10 min for PL chip; AUC₃₀, until 30 min for AR chip) under various shear rates (1000, 1500, 2000 s⁻¹ for PL chip; 300 s⁻¹ for AR chip). Platelet function of the blood sample was also tested using platelet aggregometry (collagen, ADP, ristocetin, and epinephrine as agonists), PFA-100 (C/EPI-, C/ADP-CT: closure time) and VerifyNow P2Y12 assay (PRU).

In PL chip, T₁₀ was correlated with C/EPI- and C/ADP-CT, and AUC₁₀ was correlated with C/EPI-CT under all of the shear conditions. The correlation was enhanced in accordance with the increase of the shear rates. In addition, T₆₀ and AUC₁₀ were correlated with AUC of collagen-induced aggregation curve of platelet aggregometry. In AR chip, T_10–80, reflecting the rate of thrombus growth, was likely correlated with C/ADP-CT. Measured values from VerifyNow P2Y12 assay was not significantly associated with those from this system. Interestingly, platelet numbers were significantly correlated with all of the measurements with AR chip, and partially with those with PL chip.

In healthy subjects, PTF starting time and AUC with PL chip, and the growth rate of PTF with AR chip, seemed associated with PFA-100 measurements, indicating its characteristics related to shear induced PTF. However, the values from this system showed a rare correlation with those from platelet aggregometry and VerifyNow P2Y12 assay. This system may allow us to identify the parameters of individuals' thrombogenicity independent of those related to other platelet function tests, under whole blood flow conditions.

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Typical flow pressure pattern during PTF using AR chip.

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Typical flow pressure pattern during PTF using AR chip.

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Matsubara:Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees. Ohnishi:Fujimori Kogyo Co.: Employment. Hosokawa:Fujimori Kogyo Co.: Employment. Murata:Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees.